The incorporation of tubulin into the synaptosomal plasma membrane has been recently demonstrated (Feit, PNAS, in press). In becoming membranous, most of the serine and threonine residues of the protein are glycosylated. Preliminary evidence indicates that the sugar moiety is a single species with a molecular weight of about 1000. The aim of the proposed research is to characterize the alkali-labile carbohydrate moiety in membrane tubulin and to study the distribution of the glycosylated serine and threonine residues in synaptosomal plasma membrane tubulin derived from different classes of synapses. These experiments will test a new hypothesis for the biochemical basis of synaptic specificity, namely that different classes of synapses have tubulin molecules in their membranes that are different because the same sugar side chain is distributed at different sites. Different methodologies for making peptide maps that depend on the distribution of glycosylated serines and threonines are described. These methods will be tested in model experiments comparing the distribution of glycosylated serine and threonine residues in soluble and membrane tubulin. Methods for separating muscarinic cholinergic, nicotinic cholinergic, and dopaminergic synapses will be developed and the glycosylation of membrane tubulin in these different classes of synapses will be studied. The subunit structure of tubulin will be investigated in more detail. The principal investigator has recently shown that both the alpha and beta subunits of tubulin can each be resolved into two species. The biochemical basis and the biological significance of the heterogeneity of tubulin subunits will be studied with respect to primary structure differences (peptide maps, amino acid composition), glycoscylation, phosphorylation, and potential for re-assembly into microtubules.